Various publications or patents are referred to throughout this application or at the end of this specification to describe the state of the art to which the invention pertains. Each of these publications or patents is incorporated by reference herein. Citations of scientific publications are set forth in the text or at the end of the specification.
Control of cell numbers in mammals is determined, in part, by a balance between cell proliferation and cell death. Cell death involves processes that are equal in complexity and regulation to those involved in cell proliferation. There are two forms of cell death. One form of cell death is referred to as necrotic cell death. It is typically characterized as a pathologic form of death resulting from cellular trauma or injury. Necrosis is a process that involves loss of membrane integrity and uncontrolled release of cellular contents, giving rise to inflammatory responses. In necrotic cell death, the cell has a passive role in initiating the process of death, it is a response to pathologic changes initiated outside of the cell that results in a change in the plasma membrane permeability that results in cellular edema and the osmotic lysis of the cell. In contrast, the other form of cell death, referred to as apoptosis, usually proceeds in an orderly or controlled manner wherein the cell undergoes an energy-dependent process of cellular death initiated by specific signals in an otherwise normal microenvironment (see, e.g., Barr, et al., Bio/Technology, 12:487-493 (1994); Steller, et al., Science, 267:1445-1449 (1995)).
Apoptosis, or programmed cell death, is a natural ‘physiologic’ process that occurs during growth and development, and it is an important regulator of tissue homeostasis and aging. It is the process, whereby organisms eliminate unwanted cells to prevent uncontrolled cell proliferation and or disease. Early on in development apoptosis plays a central role in sculpting the fetal animal, precisely managing cell number in tissues and controlling the formation of organs. In homeostasis, apoptosis regulates cell number, facilitates morphogenesis, removes harmful or otherwise abnormal cells, eliminates cells that have already performed their function, and hence, plays a crucial role in the development and maintenance of multicellular organisms by eliminating superfluous or unwanted cells. It further serves as a defense mechanism to remove potentially dangerous cells, including virus-infected cells, self-reactive lymphocytes in autoimmune diseases, or malignant cells and may minimize the risk of developing potentially cancerous cells in tissues frequently exposed to mutagenic chemicals, carcinogens, or UV radiation.
For instance, recent evidence has demonstrated that the rate of bone formation is regulated not only by the rate of osteoblast formation but also by the rate of osteoblast apoptosis. Thus, increased osteoblast apoptosis is at least partially responsible for the reduced bone formation in glucocorticoid excess-induced osteopenia (Weinstein, R. S., Jilka, R. L., Parfitt, A. M., and Manolagas, S. C. (1998) J. Clin. Invest. 102, 274-282). Conversely, inhibition of osteoblast apoptosis is a likely mechanism of the anabolic effect of intermittent administration of parathyroid hormone (Jilka, R. L., Weinstein, R. S., Bellido, T., Roberson, P. K., Parfitt, A. M., and Manolagas, S. C. (1999) J. Clin. Invest. 104, 439-446).
The programmed cell death process is often associated with characteristic morphological and biochemical changes. Once committed to apoptosis, cells undergo new rounds of protein synthesis and various morphological and physiological changes. The morphological characteristics of apoptosis include organelle re-localization and compaction, cell shrinkage (condensation of nuclear chromatin, nucleoplasm and cytoplasm), the appearance of membrane ruffling, plasma and nuclear membrane blebbing and formation of apoptotic bodies (membrane enclosed particles containing intracellular material), and loss of cell-cell contact followed by fragmentation. Signals for apoptosis promote the activation of specific calcium- and magnesium-dependent endonucleoases that cleave the double stranded DNA at linker regions between nucleosomes. At the end of the process, the phospholipid phosphatidylserine, which is normally hidden within the plasma membrane, is exposed on the cell's surface and bound by neighboring epitheliel cells, macrophages and dendritic cells that engulf and phagocytose the fragments from the apoptotic cell before lysis. In this way, dead cells are removed in an orderly manner without any leakage of their noxious and potentially proinflammatory contents. Because of this clearance mechanism, inflammation is not induced despite the clearance of great numbers of cells. By contrast, during the pathological form of cell death referred to above as necrosis, the mitochondria within the cell swell, lose their function and are rapidly lysed, thereby releasing cytoplasmic contents that invariably trigger an inflammatory response.
In general, the apoptosis cycle can be divided into three phases: an initiation phase in which the various death stimuli take so-called “private” pathways to converge on a common effector phase involving the caspase family of proteins, which leads finally to the degradation phase characterized by the typical biochemical symptoms of cell death. In phase one, the cell undergoes genetic reprogramming in which certain genes that were previously expressed are now repressed, while other genes that were previously repressed are now expressed. These genetic changes result in the activation of double-stranded DNA fragmentation during the next phase.
During phase two, the effector phase, the nuclear morphology changes (i.e., nuclear condensation of chromatin), while the plasma and lysosomal membranes remain intact and the mitochondria continue to function. This phase is regulated by the mitochondrial permeability transition pore since its open or closed conformations determine the fate of the cells. It participates in regulating the level of calcium, the pH and the transmembrane potential in the mitochondria. It has been demonstrated that opening of the pore, regulated by Bcl-2, is a critical event in the process leading to apoptosis as it allows dissipation of the transmembrane potential, disrupting the integrity of the outer membrane and leading to the release of mitochondrial intermembrane proteins, such as cytochrome c (Kroemer et al. 1997).
Subsequently in phase three, proteases are activated, like proteases that hydrolyzes poly(ADP-ribose) polymerase, the lamins in the nuclear membrane are degraded and the nucleus itself undergoes fragmentation. Plasma membrane blebbing and eventual cellular fragmentation into clusters of membrane-bound apoptotic bodies then occurs. Once formed, these apoptotic bodies are rapidly recognized, phagocytized, and digested by macrophages or by adjacent epithelial cells.
Once initiated apoptosis leads to a cascade of biochemical and morphological events that result in irreversible degradation of the genomic DNA and fragmentation of the cell. Entry into this programmed cell death pathway is regulated by a careful balancing act between those specific gene products that promote and those that inhibit apoptosis. A cell activates its internally encoded suicide program as a result of either internal or external signals. In a healthy cell, the protein Bcl-2 is expressed on the outer membrane surface of mitochondria and bound to an Apoptosis Activation Factor-1 (Apaf-1) protein. When a cell undergoes internal damage, Bcl-2 is caused to release Apaf-1 resulting in the disruption of the mitochondria membrane and the leakage of both Apaf-1 and cytochrome c out of the mitochondria. Once released cytochrome c and Apaf-1 bind to a caspase 9 protease to form a complex called an apoptosome, which aggregates in the cytosol. Caspase 9 is thus activated, which in turn activates other members of the caspase family of proteases which results in the digestion of the structural proteins of the cytoplasm, the degradation of chromosomal DNA and ultimately in the phagocytosis of the cell.
Endogenous activation of apoptosis occurs due to the positive presence of a tissue-specific external signal (such as TNF-α or glucocorticoids) that induces cells to self-destruct. One characteristic of the tumor necrosis factor (TNF) family is the ability of many family members to induce programmed cell death in a variety of cells, both normal and of tumor origin (Wiley et al, Immunity 3:673-682, 1995, and references therein). TNF is a cytokine that has been implicated in cell death. There are two forms of TNF, they are the α and β forms. TNF-α is a soluble homotrimer of 17 kD protein subunits. A membrane-bound 26 kD precursor form of TNF-α also exists. TNF α and β are produced from various cells, including, for example, T cells, monocytes, macrophages, and natural killer cells, by induction with prophiogistic agents such as bacteria, viruses, various mitogens or the like.
TNF elicits a broad range of biological effects through two distinct membrane receptors, TNF R1 and TNF R2, which are expressed at low levels on most cell types. Endotoxins strongly activate monocyte/macrophage production and secretion of TNF. It is a mediator of the metabolic and neurohormonal responses to endotoxins. TNF causes the pro-inflammatory actions that result from tissue injury, increases the adherence of neutrophils and lymphocytes, and stimulates the release of platelet activating factor from macrophages, neutrophils and vascular endothelial cells. To this extent, TNF is a key component in biological activities such as inducing hemorrhagic necrosis in tumors, apoptosis in cancer cells, production of prostaglandins and collagenase, expression of adhesion. molecules (ICAM-1, ELAM-1) and HLA class II molecules, production of inflammatory cytokines (e.g., IL-I, IL-6) and chemokines (IL-8, RANTES), and enhancement of absorption of bone and cartilage.
One of the most striking features of TNF compared to other cytokines is its ability to elicit programned cell death. Apoptosis induced by TNF is mediated primarily through TNF R1. The intracellular domain of TNF R1 contains a “death domain” of approximately 80 amino acids that is responsible for signaling cell death by the receptor. When TNF a binds to its integral membrane receptor (TNF R1) a signal is transmitted to the cytoplasm activating caspase 8, which initiates an expanding cascade of sequential caspase family activation and proteolytic activity that results in the eventual phagocytosis of the cell.
Along those lines, it was determined that the inflammatory cytokine TNF, which inhibits bone formation, collagen synthesis, and alkaline phosphatase (Stashenko, P., Obernesser, M. S., and Dewhirst, F. E. (1989) Immunol. Invest. 18, 239-249; and Centrella, M., McCarthy, T. L., and Canalis, E. (1988) Endocrinology 123, 1442-1448), also induces apoptosis of osteoblastic cells (Jilka, R. L., Weinstein, R. S., Bellido, T., Parfitt, A. M., and Manolagas, S. C. (1998) J. Bone Miner. Res. 13, 793-802; Hill, P. A., Tumber, A., and Meikle, M. C. (1997) Endocrinology 138, 3849-3858; and Kitajima, I., Nakajima, T., Imamura, T., Takasaki, I., Kawahara, K., Okano, T., Tokioka, T., Soejima, Y., Abeyama, K., and Maruyama, I. (1996) J. Bone Miner. Res. 11, 200-210). However, it was further determined that growth factors such as insulin-like growth factor I, basic fibroblast growth factor, interleukin-6 type cytokines, and transforming growth factor inhibit osteoblastic cell apoptosis induced by TNF, serum deprivation, or activation of Fas (Jilka, R. L., Weinstein, R. S., Bellido, T., Parfitt, A. M., and Manolagas, S. C. (1998) J. Bone Miner. Res. 13, 793-802; Hill, P. A., Tumber, A., and Meikle, M. C. (1997) Endocrinology 138, 3849-3858; and Kitajima, I., Nakajima, T., Imamura, T., Takasaki, I., Kawahara, K., Okano, T., Tokioka, T., Soejima, Y., Abeyama, K., and Maruyama, I. (1996) J. Bone Miner. Res. 11, 200-210).
Interestingly, insulin growth factor I and fibroblast growth factor induce the calcium binding protein calbindin-D28K expression in neurons and promote neuronal survival (Collazo, D., Takahashi, H., and McKay, R. D. (1992) Neuron 9, 643-656; Nieto-Bona, M. P., Busiguina, S., and Torres-Alemañ, I. (1995) J. Neurosci. Res 42, 371-376; Mattson, M. P., Murrain, M., Guthrie, P. B., and Kater, S. B. (1989) 1 Neurosci. 9, 3728-3740; and Cheng, B. and Mattson, M. P. (1992) J. Neurosci. 12, 1558-1566). Recent evidence suggests the involvement of apoptosis in the regulation of osteoblastic bone formation and osteoclastic bone resorption during adult bone remodeling (Hughes, D. E., and Boyce, B. F. (1997) J. Clin. Pathol (Lond.) 50, 132-137; and Manolagas, S. C. ((1999) Endocrinology 140, 4377-4381). Because of these facts the inventor set out to determine whether expression levels of calbindin-D28K had an effect on apoptosis of osteoblastic cells. The results showed TNF induced nuclear fragmentation of MC3T3-E1 cells transfected with an empty vector, but that this pro-apoptotic effect of TNF was significantly attenuated in cells transfected with calbindin-D28K cDNA. Hence, it was determined that calbindin-D28K transfectants were resistant to TNT-induced apoptosis.
Since some evidence indicated a role for calcium in the initiation as well as in the degradation phase of apoptosis, it was suggested that the anti-apoptotic effect of calbindin-D28K could be due to its ability to chelate calcium. It was also suggested that calbindin-D28K could inhibit the release of cytochrome c from the mitochondria, which is needed for the activation of caspase-3, by preventing calcium mediated apoptotic damage of mitochondrial electron transport (Guo, Q., Christakos, S., Robinson, N., and Mattson, M. P. (1998) Proc. Natl. Acad. Sci. U S. A. 95, 3227-3232). Because recent evidence indicates that the caspase family of proteins play a role in inducing TNF evoked apoptosis it was suggested that calbindin-D28K may interact with one or more of the caspase family of proteases to inhibit apoptosis.
The caspases, are a family of cysteine proteases that share the characteristic feature of a conserved QAC(R/Q)G motif (SEQ ID NO:3), in which the Cys residue is part of the active site and is essential for caspase-mediated apoptosis. These proteases are primarily responsible for the degradation of cellular proteins that lead to the morphological changes seen in cells undergoing apoptosis. It has been shown that many members of the family are capable of inducing apoptosis when overexpressed in mammalian cells (Henkart, 1996 and Miura et al., Cell 75:653 [1993]). For instance, it is known that caspase 1 (interleukin 1β converting enzyme or ICE) is responsible for the activation of interleukin-1β (IL-1β) and is necessary for apoptosis. It is a substrate-specific cysteine protease that cleaves the inactive prointerleukin-1 to produce the mature IL-1. IL-1 is a cytokine involved in mediating a wide range of biological responses including inflammation, septic shock, wound healing, hematopoiesis and growth of certain leukemias and apoptosis. When caspase 1 is overexpressed cell apoptosis can be induced. Over expression of caspases 2 and 3 in fibroblasts and neuroblastoma cells also results in cell death by apoptosis.
Caspase 3 is a protein also known to be intimately involved with a cell's ability to induce apoptosis in normal nuclei. Caspase 3 normally exists in the cytosolic fraction of cells as a 32 kDa inactive precursor that is converted proteolytically to a 20 kDa and a 10 kDa active heterodimer when cells are signaled to undergo apoptosis (Schlegel, et al., Biol. Chem. 271:1841-1844, (1996); Wang, et al., EMBO J. 15:1012-1020, (1996)). And it is known that Bcl-2, prevents the activation of caspase-3 by blocking the mitochondria from releasing cytochrome c, a necessary co-factor for caspase-3 activation (Liu, et al., Cell 86:147-157, (1996); Yang, et al., Science 275:1129-1132, (1997); Kluck, et al., Science 275:1132-1136, (1997)).
Another extrinsic signal that can trigger apoptosis comes from glucocorticoid hormones. Glucocorticoids exert several effects in tissues that have receptors for them. By binding to their receptors, glucocorticoids regulate the expression of several genes either positively or negatively, in a direct or indirect manner, and are known to arrest cell growth and can induce cell death.
The human glucocorticoid receptor is made up of 777 amino acids and is predominantly cytoplasmic in its unactivated, non-DNA binding form. When activated, it translocates to the nucleus. There are four major functional domains of the glucocorticoid receptor. The first, from the amino terminal, is the tau I domain, which spans amino acid positions 77-262 and regulates gene activation. The second is the DNA binding domain, which spans amino acid positions 421-486 and contains nine cysteine residues, eight of which form two different zinc fingers. The DNA binding domain binds to the regulatory sequences of genes that are induced (or deinduced) by glucocorticoids. The tau 2 domain runs from amino acids 532-555 and it is also important for transcriptional activation. Towards the carboxyl terminal end, from amino acids 555 to 777, is the steroid binding domain, which binds glucocorticoid to activate the receptor. This region of the receptor also has the nuclear localization signal.
The glucocorticoid receptor is expressed in the cytoplasm of a cell. When a glucocorticoid enters a cell and binds its receptor, the receptor goes through a conformational change wherein it is activated, forms a heterodimer with another glucocorticoid bound receptor. complex and is transported via a transport protein to the nucleus of the cell. This heterodimer complex interacts with the cell's DNA upregulating the production of the pro-apoptotic signal:bax. The bax protein is transported to the surface membrane of a mitochondria wherein it forms a pore, causing the release of cytochrome c, which then binds to the Apaf-1 protein resulting in the activation of the caspase cascade and phosphatidyl serine being displayed, which leads to the engulfment and degradation of the cell by neighboring epithelial and macrophage cells.
As stated above, signals that initiate apoptosis trigger the so-called private pathways of death, which are specific for particular groups of stimuli and lead to the conversion of procaspases to active the caspases. Although both TNT a and the glucocorticoid hormones can induce apoptosis by binding to their respective receptors and activating the caspase family cascade, little is known about the actual signaling events that occur once the caspases are activated, nor what caspases are activated and by which inducer, nor which lead to the common cell death pathway. The end result of this cascade, however, is chromatin condensation, nuclear fragmentation, increase in cell membrane permeability, and ultimately cell death (Thornberry, N. A., and Lazebnik, Y. (1998) Science 281, 1312-1316; and Green, D. R. (1998) Cell 94, 695-698).
The inventor's previous work establishes a relationship between calbindin-D28K and caspase-3 in that it was determined that calbindin-D28K could prevent TNF induced apoptosis by interacting with caspase-3 via an unknown mechanism. (See Bellido et al. (2000) Journal of Biol. Chem. 275, 26326-26332.) The present work of the inventor, and the subject matter of the present invention establishes that calbindin-D28K can also prevent glucocorticoid induced cell death. Given that apoptosis is tightly regulated and has been linked to pathways that are dysregulated in a variety of diseases including cancer, the present invention is important because it further elucidates a mechanism by which to control this process, especially as it relates to glucocorticoid induced cell death. For instance, the compositions and methods of the present invention are useful in the prevention and treatment of glucocorticoid induced osteoporosis, which is the third most prevalent form of osteoporosis after postmenopause and senile osteoporosis.